The last decade has witnessed a decline in the resources of natural fisheries that has now reached a level of global environmental crisis. Thirteen of the worlds seventeen major fisheries are classified by the United Nations Food and Agriculture Organization (F.A.O.) to be in peril or in steep decline. Even in the best case scenario of both maintaining the fishery catch at a record 100 million metric tons and conservative population growth, there will be a shortfall in global seafood supply by the end of this decade for the first time in human history. Furthermore, per capita global seafood consumption is predicted to increase as the worlds population shifts during the next two decades from industrialized countries to todays undeveloped and emerging economies; traditionally large consumers of seafood. The only way to meet the global demand for seafood by the year 2000 is if aquaculture replaces the insufficient natural fisheries catch.
Expansion of the aquaculture industry requires that several significant problems be addressed, and one of the most significant hurdles to establishing and maintaining an economically viable aquaculture operation is the difficulty of supplying nutritionally balanced feeds. Larval fish, bivalves and crustaceans raised in the wild consume a mixed population of feed organisms that collectively provide balanced nutrition. On the other hand, fish larvae, bivalves and crustaceans raised in aquaculture farms can be difficult to rear and require live feeds (algae or algae-fed rotifers and Artemia) for their nutrition. These live feeds are difficult to produce and maintain, require high labor inputs and specialized facilities, and as a result larval feeds constitute a significant cost to the aquaculture industry.
It is important that aquaculture feeds be nutritionally balanced so that the larvae receive proper nutrition. DHA (docosahexaenoic acid) has been identified as an important nutrient that contributes significantly to larval growth and survival (Fulks, W and KL Main (eds). 1991. xe2x80x9cRotifer and Microalgae Culture Systems. Proceedings of a US-Asia Workshop.xe2x80x9d Honolulu, Hi. The Oceanic Institute. Tamaru, CS, CS Lee and H Ako, 1991. xe2x80x9cImproving the larval rearing of striped mullet (Mugil cephalus) by manipulating quantity and quality of the rotifer, Brachionus plicatilis.xe2x80x9d In: W. Fulks and KL Main (eds), 1991). Larvae ultimately acquire these fatty acids from algae, either by directly feeding on algae with high levels of polyunsaturated fatty acids or by feeding on rotifers and Artemia that have been reared on algae high in polyunsaturated fatty acids. Unfortunately, the algae, artemia and rotifers used at aquaculture farms are low in DHA, reducing the survival rates for the larvae below their maximal rate and increasing the cost of the final aquaculture farm product. If sufficient DHA could be provided to the larvae it is expected that the survival rate for larvae would increase, thus reducing the cost of farm-raised seafood.
In one embodiment, this invention provides a particulate material suitable for use as an aquaculture feed. The particulate material has a high proportion of DHA residues in the lipid fraction, which may be up to 35% of the material. Preferably, the material has a mean particle size of from about 5 microns to about 10 microns.
In another embodiment, this invention provides a method for preparing a particulate material suitable for use as an aquaculture feed from microbial biomass, preferably from algal cells having a high content of DHA residues, by obtaining a lipid fraction from the biomass, preferably by solvent extraction of broken cells, followed by separating a fraction containing phospholipids and proteins from the lipid fraction, and removing water from the protein/phospholipid fraction to form a low moisture particulate, preferably by spray-drying the protein/phospholipid fraction.
In still another embodiment, this invention provides a particulate material containing phospholipids having docosahexaenoic acid (DHA) residues, the particulate material being prepared by drying a slurry comprising a polar lipid extract from DHA-containing microbes, and dry, particulate material may be prepared from a slurry which is substantially free of material that did not come from the DHA-containing microbes, particularly by spray drying the slurry. Typically, at least two thirds of the dry matter in the polar lipid extract of DHA-containing microbes is material derived from the microbial cells; preferably less than 25% of the dry matter is non-microbial, more preferably less than 20%, even more preferably 15% or less. Typically, the spray dried particulate has number average particle size between 5 microns and 10 microns.
In yet another embodiment, this invention provides a method for preparing a DHA-containing particulate material comprising (a) lysing DHA-containing microbial cells; (b) extracting lysed cells with solvent; (c) separating a polar lipid fraction from the extract; and (d) drying the polar lipid fraction, with or without addition of other nutrients, to form a particulate material. Preferably, the polar lipid fraction is an aqueous slurry which is dried by spray drying.
In a particular embodiment, this invention provides a method for preparing a DHA-containing particulate material comprising drying a slurry containing polar lipids extracted from dinoflagellates, wherein the dried material is in the form of particles having a mean particle diameter between 5 and 10 microns.
In yet another embodiment, this invention provides an aqueous emulsion or suspension containing phospholipids with DHA residues obtained from a polar lipid extract from DHA-containing microbes. Preferably, at least 10% of the fatty acid residues in lipids of the DHA-containing microbes are DHA residues. More preferably, at least 10% of the fatty acid residues in polar lipids of the DHA-containing microbes are DHA residues.
In still another embodiment, this invention provides a composition comprising a particulate material containing phospholipids with DHA prepared by drying a slurry comprising (a) a polar lipid extract from DHA containing microbes and (b) a meal containing protein and/or carbohydrate. Preferably, the meal comprises microbial cells or cell fragments, which may be cells or cell fragments which have been extracted to remove part of the lipids, or even most of the lipids. Preferred microbial cells or cell fragments are from Chlorella, Crypthecodinium, or a yeast such as Saccharomyces, or a fungus, such as Morteriella, Schizochytrium, or Thraustochytrium.
Producing high DHA-containing oil from biomass requires several different processing and refining steps. Each refining step produces waste fractions as a byproduct of the oil purification. These byproducts contain significant levels of DHA and other nutrients that can have potential use as aquaculture feeds for fish larvae, bivalves and crustaceans.
One byproduct in particular contains a very high level of DHA as well as other nutrients (carbohydrate, protein, etc) that are useful and valuable in larval nutrition. This byproduct, hereinafter called xe2x80x9cDHA Phospholipids,xe2x80x9d also contains significant levels of all of the amino acids that are considered essential for larvae. The ten amino acids that are generally recognized to be essential for most fish species are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, see, e.g., Shepherd, Jonathan and Niall Bromage, Intensive Fish Farming, Chapter 5, Nutrition and Growth, Blackwell Scientific Publications, Boston, 1992, pp. 154-197. The DHA Phospholipid material contains all of these amino acids at levels comparable to the levels found in some other aquaculture feeds. The DHA Phospholipid material contains phospholipids having a high proportion of DHA residues, as well as other lipid components such as triglycerides, free fatty acids, sterols, glycolipids, etc. The biochemical composition of the DHA Phospholipids, obtained as spray-dried particles, is given in Table 1. The fatty acid composition of the particulate material is given in Table 2.
Source of DHA-containing Material
The principal source of DHA in the biosphere is from algae. Numerous algal species produce DHA. One species in particular (Crypthecodinium cohnii) has been manipulated to produce very high levels of DHA. This organism has been cultivated at large scale and the biomass has been used for the production of a high DHA-containing oil. Suitable processes for producing DHA-containing biomass from C. cohnii are provided in U.S. Pat. Nos. 5,397,591 and 5,492,938, incorporated herein by reference.
Similar DHA Phospholipid material may be made from any single celled organism that contains a significant amount of DHA This would include various oleaginous fungi, various algae (especially members of the class Dinophyceae, Bacillariophyceae, Chlorophyceae, Prymnesiophyceae and Euglenophyceae), as well as organisms of uncertain taxonomic status such as Thraustochytrium or Schizochytrium. Suitable processes for producing DHA-containing biomass from Thraustochytrium or Schizochytrium are provided in U.S. Pat. No. 5,130,242, incorporated herein by reference.
Typically, microbes serving as a source of DHA Phospholipids according to this invention will have at least 5% of the fatty acids and fatty acid residues in the microbe as DHA, more typically at least 10%, and preferably at least 20%. Preferably, at least 10% of the fatty acid residues in the polar lipids of the source microbe will be DHA, more preferably at least 20% of the residues will be DHA.
Preparation Methods
DHA Phospholipids may be obtained from a lipid fraction of the DHA-containing biomass. A preferred method for obtaining a suitable lipid fraction is solvent extraction of lysed cells. In a preferred mode, the biomass may be spray-dried, which will lyse most cells, and the spray-dried material extracted with a solvent such as hexane. Grinding or homogenizing the solvent-biomass mixture may facilitate extraction of the lipid fraction (oil) and may also promote recovery of non-lipid components that are desirable for the aquaculture feed. The DHA Phospholipid material is separated from the oil as a water/salt-containing precipitate. Suitable procedures for such separation are readily adapted from analogous processes in vegetable oil refining.
Although the DHA Phospholipid byproduct contains useful levels of DHA and other nutrients, a suitable method must be found to provide this material to larvae. Many fish larvae require live feed, and bivalves and crustaceans also require either live feed organisms or particles of a limited size range. Thus, further processing is preferable to merely providing this material directly to the fish larvae, bivalves or crustaceans.
For those organisms requiring a live feed, the DHA Phospholipids can be fed to arterria and/or rotifers (both natural food organisms for larvae), with the goal of raising the DHA level of the artemia/rotifers and subsequently raising the DHA level of the larvae. For those organisms that are filter feeders, the material may be supplied as small particles and thus provide a direct enrichment of DHA into the diet.
In order to prepare particles of the proper size for ingestion by artemia, rotifers and filter feeders, the DHA Phospholipid material is dried by any suitable process, such as spray drying, granulation, encapsulation and the like. When DHA Phospholipid material was freeze dried, the resultant particles were too large to be useful. The high fat content in the freeze-dried material did not permit the particle size to be reduced by grinding. Spray drying yielded a crumbly mass consisting of particles that were of an appropriate size range for consumption by artemia, rotifers and filter feeders. Mean particle size was determined to be about 5-10 microns in diameter; the distribution of partical sizes about the mean was quite broad ( at least from  greater than 30 microns to  less than 1 micron). Although the material was subjected to heat during the drying process, the DHA level was not appreciably reduced by the temperature of the dryer. Thus, spray drying yielded a product with a high DHA content and an appropriate particle size.
Preferably, the DHA level of the spray dried material should be maximized, thus making the material an even more valuable source of DHA for larvae. Typically, the DHA level would be raised by increasing the amount of DHA-containing oil in the DHA Phospholipid material, which may be accomplished by adding refined triglyceride oil, partially purified triglycerides or crude lipid extract from a microorganism that is high in DHA. The inventor has noted that, when the triglyceride level (measured as esterified fatty acid level) exceeded about 30% of the total dry weight of the DHA Phospholipid, the material did not spray dry as well and appeared to form very large particles that were less suitable for this application. At triglyceride contents above 40%, the final spray-dried product is very sticky due to the high fat content, which may explain the apparent high particle size. This material is not as easy to handle as the lower triglyceride material, but it still can be used as an artemia and rotifer enrichment product. Of course, supplemental DHA may also be supplied from non-microbial sources, including purified DHA-alkanol esters, but these sources may be less economical.
While DHA Phospholipid may be spray dried directly, spray drying may be facilitated if the slurry contains typical spray drying agents when it is fed into the spray dryer. Suitable spray drying carriers include maltodextrines, starch, gelatin, sugars, and molasses. Preferred carriers include sodium alginate and gum arabic. In addition, various additives whose presence in the final particulate is desirable for nutritional reasons may also be added to the slurry before drying, such as proteins, carbohydrates, fatty acids and lipids, or vitamin sources, including additives like yeast extract, starch, gelatin, gluten, etc. In addition, amino acids may be added to the slurry, such as methionine, a particularly important amino acid in fish nutrition. Alternatively, algal biomass may be added to the slurry as a spray drying aid, preferably using algae which are naturally food for rotifers or fish larvae.
In one embodiment, particulate material may be prepared by formulating and spray drying the DHA Phospholipids with microbial biomass, which may be whole or lysed cells or cellular material after lipid extraction. DHA Phospholipids may be mixed with biomass in any ratio, including 1:100 to 100:1, preferably in ratios from 1:10 to 10:1. The mixture is typically spray dried from an aqueous slurry to form a material with the appearance or consistency of cornmeal. Typically, the biomass will be from microbes that have known nutritional uses, such as Chlorella. Chlorella is consumed as a human food product, and DHA is an important human nutrient, so such particulate material is also useful in human nutrition. Of course particulate material produced according to this invention may be used to supply DHA in human nutrition whether or not the particulate contains Chlorella, so long as the process conditions and any other additives included in the slurry are suitable for human consumption.
While the DHA Phospholipid is particularly suited to spray drying, particulate material containing high DHA levels may also be prepared according to this invention by granulation or encapsulation. Conditions for granulation or encapsulation of DHA Phospholipid may be readily selected by the skilled worker, typically using one or more of the additives which are listed above for use in spray drying, such as alginate or gum acacia. Of course, DHA Phospolipids may also be coated on oilseed meal (e.g., soybean meal, cotton seed meal, corn germ, etc.). Furthermore, mixtures of DHA Phospholipids with solvent extracted algal biomass (xe2x80x9cbiomealxe2x80x9d) are also suitable for freeze-drying. While this invention is particularly suitable for producing dry products enriched in DHA, the DHA Phospholipids obtained from microbes according to this invention may be formulated in a suspension or emulsion for subsequent use, particularly as a nutritional supplement. Such emulsions or suspensions containing polar lipids may be routinely prepared, as is known in the art.
Particle size distribution in the dried material may be adjusted by fractionation in, e.g., cyclones or filter bags, to remove particles that are too large, but preferably the drying process will produce a particulate material of mean particle size from 5 microns to 10 microns. Typically the dried DHA Phopholipids will be mixed with sea water before feeding to larvae, rotifers, or artemesia. Varying the agitation in this mixing step will permit post-drying adjustment of the particle size.
Another fatty acid with significance in both aquaculture and human and animal nutrition is arachidonic acid (AA). Arachidonic acid may also be obtained in lipid extracts of microbes, although preferred sources of AA are different species from the preferred sources of DHA. Suitable microbial sources of AA and methods for growing them are taught, for example, in U.S. Pat. No.5,658,767. Extraction of crude lipids from AA-containing microbes leaves a protein-and-carbohydrate containing residue that may also still have appreciable amounts of AA as fatty acids and/or fatty acid residues. This material may be referred to as AA biomeal.
During the processing and refining of the AA-containing crude lipid extract, several fractions are produced, including AA Phospholipids. The AA Phospholipids can also be blended with the DHA Phospholipids to produce a material that contains both fatty acids in the ratio that will provide for optimal nutrition. Such a blend can be prepared as a dry material or as an emulsion or suspension. The AA Phospholipids can also-be blended with the DHA biomeal and dried or emulsified to produce a material that is high in arachidonic acid. Through the use of different ratios of DHA Phospholipids, DHA biomeal, AA biomeal, and/or AA Phospholipids, a material can be produced that has the appropriate ratio of DHA and AA for optimal nutrition.